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Microfluidic technology for efficient and high yield gene delivery

The first step in many genetic related studies is the transfection that is the process of delivering a genetic cargo into the cells. The main challenge for both therapeutic and diagnostic use of genetic methods such as CRISPR-Cas is the low transfection rate. The cell is designed such that it does not allow unwanted external materials to enter the cell. Therefore, it is not trivial to deliver genetic material to the cell. The cell can either voluntarily uptake the external material via endocytosis or be forced to absorb the external material. In the former, the genetic material is carried by vesicles in a way that the cell does not identify it as a predator and devours the vesicle, and therefore the genetic material. An example of this is cationic lipid lipofection. In the latter technique, the membrane of the cell is rendered permeable via an external force. This external force could be an electric voltage (electroporation), ultrasound wave (sonoporation), magnetic force (magnetofection), etc. Each of them has its own advantages and disadvantages and can be fit for a specific condition. The challenge is that even with the external force, the transfection rate barely reaches 10%. The low transfection rate is a significant barrier for researchers aiming at delivering novel therapeutic and diagnostic techniques to the market. Microfluidics approaches for gene delivery, however, can be of help. 

Microfluidics technology is well-known for its capacity of handling very small volumes of liquids within micron-scale confinements. Bulk methods rely on random exposure of the genetic material to the cells in a relatively large area. In microfluidic devices, however, the large surface to volume ratio increases the chance of interaction between the genetic material and the target cell. This microfluidic approach can dramatically increase the transfection rate.

Microfluidics lipofection methods for cell transfection

Lipofection is associated with using a lipid material as a carrier for delivering the DNA. The lipid is attached to a tail with a positive charge. The lipids then self-assemble to form liposomes and attract the DNA with the opposite charge. The cell membrane is made of a phospholipid bilayer and can easily merge with the liposome. The nucleic acid cargo of the liposome vesicle then gets released inside the cell. The bulk techniques for lipofection normally have a very low success rate. Microfluidic lipofection techniques, however, have been able to increase the transfection rate by one order of magnitude.

lipofectionA popular microfluidic chip based lipofection approach is droplet microfluidics. In droplet microfluidics, a droplet generator chip is used to generate droplets of micron size. Depending on the application, the droplets can be water in oil droplets or oil in water droplets. For lipofection, normally, the droplets are water in oil droplets. In order to make water in oil droplets, two different phases are required: The oil phase or the carrier phase and the aqueous phase. The aqueous phase carries the cells, lipids, and the genetic material and eventually pinches off into droplets. The cells+plasmid solution and the lipofectin solution are introduced to the device via separate inlets. Next, they meet and form a homogeneous aqueous phase that burst into droplets by the oil phase. It is recommended to use fluorinated oil such as FC40 since hydrocarbon oils like mineral oil. Higher gas permeability and low solubility for organic materials in fluorinated oils lead to higher cell viability and a lesser chance of losing the genetic materials.

The aim is to encapsulate a single-cell in each droplet. However, because of the Poisson distribution of the randomly distributed cells, some droplets will end up being empty. Increasing the concentration of the cells can lower the number of empty droplets but will also result in some droplets having more than one cell. Upon droplet formation, the lipids start to aggregate and attract the negatively charged DNA to form liposome carriers. The small droplet volume dramatically increases the probability of the liposome to be endocytosed. The microfluidics droplet formation can be followed by passing through a mixing channel where the droplets are agitated to enhance the internal flows within the droplets. More chaotic flows inside the droplets increase the transfection rate. The droplets can then collapse by shaking to release the cells for further analysis.

Microfluidic electroporation approaches for delivering genetic and chemical cargo

Electroporation refers to the process of breaching the cell membrane by using an external electric field. The electric field temporarily makes the cell membrane permeable allowing materials to infuse into or diffuse out of the cell. Electroporation is used to either introduce chemicals and genetic materials to the cell or extract metabolites. Conventionally, the cells are immersed in a chamber and between two parallel electrodes. The chamber size varies in the range of mm to cm. As soon as the electric field applies, the cell membrane loosens allowing cargo transfer between the solution and the cell. The gap between the electrodes is significantly larger than the cell size. This large gap necessitates a high voltage for effective electroporation. Applying a high voltage can cause Joule heating, change pH, or form bubbles. These side effects can negatively affect cell viability and decrease the electroporation efficiency. The need for high voltage can easily be overcome by using microchannels. Microfluidics Fabrication processes allows precise control of microenvironments within a bio-compatible material and is well-suited for cellular assays. The microchips can simply be integrated with electrodes to transfect cells in micron-sized channels. The small channel size eradicates the need for high voltages resulting in higher cell viability. As opposed to the bulk method, the cells are not immersed in a large area. Therefore, they experience a more uniform electric field that leads to more uniform membrane poration.Electroporation-on-a-chip

Electrodes implemented in microfluidic electroporation chips vary depending on the application. A simple way is to sandwich the microfluidic chip between two parallel electrodes. Applying the voltage to the plates will then permeabilize the cell membrane allowing the cargo transfer into and out of the cells. Parallel electrodes will make a uniform electric field but they block the view. Thus, parallel electrode plates are not a good option for real-time analysis.

Coplanar electrodes are another type of common form of electrodes. They are more versatile and are suitable for both adherent and suspended cells. Coplanar electrodes normally sit on the bottom of the channel. Therefore, they do not block the top-view and can be in direct contact with the chemicals. These electrodes are made via the lift-off process. This allows a wide variety of electrodes to be designed and manufactured based on the need. Coplanar electrodes range from two stripes of electrode printed on the bottom surface (typically a glass slide) to high-density interdigitated electrodes. Increasing the number of electrodes reduces the voltage required for successful electroporation. More caution is needed to make sure the generated electric field since the field is limited to the surrounding of the electrodes. A rule of thumb for designing coplanar electrodes is to make sure the distance of the electrodes should be larger than the microfluidic channel height. 

What are the advantages of using microfluidic technology for gene delivery?

Molecular biology methods can benefit from parallel techniques for gene delivery. Microfluidic chips are easily parallelizable to increase throughput. As opposed to conventional techniques such as microarrays that require a significant amount of labour work, microfluidic chips can be automated to reduce the preparation and operation time.

Conventional benchtop techniques are associated with very low efficiencies and cell viability. Microfluidics channels have a high surface to volume ratio that is an important factor in increasing transfection efficiency. Microchips offer a highly controlled environment for the growth and transfection of the cells. This sterile and supervised condition ensures low cell mortality during growth, proliferation, or transfection in Microfluidic devices. Moreover, due to these highly supervised conditions., the risk of external contamination or cross-contamination drops significantly.

Last but not least, oftentimes, the convention in gene delivery and gene therapy research is to analyze the cells at the endpoint. However, microfluidics chips are easily integrated with sensors and imaging devices that enable the researcher to observe the cells dynamically. The alterations and the response of the cells to the genetic cargo can be monitored in real-time owing to the versatility of these microfluidics lab-on-a-chips.

What are the applications of gene delivery?

Many of the diseases are caused by malfunctioning a gene or group of genes. Theoretically, fixing the damaged gene must help in treating the disease. This includes delivering an exogenous genetic cargo to the cell without damaging the cell to replace or repair the mutated or missing gene. However, delivering external material to the cell is a major obstacle since the cell is not willing to accept unwanted and unknown materials.

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